Investigating the specificity of real-time PCR assays using synthetic oligonucleotides

Potato spindle tuber viroid (PSTVd) causes damaging diseases of solanaceous crops and is a quarantine pathogen in the European Union. Previously a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed and shown to be ideally suited to PSTVd detection. However, since it was impossible to trace infected plant material for every published PSTVd sequence reported, in silico predictions were made about assay specificity based on the positions of nucleotide polymorphisms within the published viroid sequences and the regions of the primers and probe. The predictions could not be verified due to the absence of viroid material. This paper describes work investigating the detection of these sequence variants by designing synthetic oligonucleotides to sequences from the database and testing them with a real-time PCR assay. The results show that all PSTVd sequence variants are detected, and that the closely related Mexican papita viroid is also detected, although with a lower efficiency. The paper gives indications as to what effect nucleotide changes at different positions within primers and probes might do and should aid in the testing of future assays, although it is difficult to draw fixed rules about the possible effect changes may have.